The long-term goal of our research is to understand the cellular mechanisms underlying cerebral ischemic damage and provide insights for development of effective treatment for stroke. Our previous studies demonstrate that a Na-dependent Cl transporter plays an important role in disruption of ion homeostasis in cerebral ischemia and contributes to cytotoxic edema and ischemic damage. In this study, we will investigate the role of Na/H exchanger isoform 1 (NHE1) in ischemic damage. It is unknown whether NHE1 activity contributes to ion perturbation in cerebral ischemia and whether inhibition of NHE1 is neuroprotective against ischemic brain damage. Our preliminary study revealed that NHE1 is essential in regulation of pHi in astrocytes and neurons. Either pharmacological inhibition or genetic ablation of NHE1 activity delayed pHi regulation and significantly reduced Na+ accumulation in astrocytes and neurons following in vitro ischemia (oxygen and glucose deprivation, OGD). Moreover inhibition of NHE1 with a potent inhibitor HOE 642 reduced infarct volume by 40% following focal ischemia. The cellular mechanisms underlying this protection are not well understood. Therefore, an extensive study of NHE1 in cerebral ischemia is warranted. We hypothesize that NHE1 activity in astrocytes and neurons is stimulated following focal ischemia. Excessive stimulation of NHE1 will lead to the intracellular Na+ overload, which in turn can cause the rise of intracellular Ca2+ (through reduced Ca efflux and/or increased Ca influx via the reversed Na/Ca exchange). Thus, NHE1 activity contributes to cerebral ischemic damage in part by disruption of intracellular Na+ and Ca2+ homeostasis. These hypotheses will be tested by three Aims: 1. Determine the role of NHE1 in pHi regulation, intracellular Na+ and Ca2+ overload, swelling, and cell damage in astrocytes following OGD. 2. Investigate the contribution of NHE1 to ischemic neuronal death. 3. Investigate whether pharmacological inhibition and genetic ablation of NHE1 reduces brain damage in mice following transient focal ischemia.